The general goal of these studies is to investigate structural and molecular mechanisms regulating the activation of factor IX via the tissue factor pathway of coagulation and to further delineate the functions of different domains of factor IX. Tissue factor pathway inhibitor (TFPI) consists of three kunitz-type domains. The first domain binds to factor VIIa and the second domain binds to factor Xa. The function of the third domain is as yet not clear. In the proposed studies, attempts will also be made to delineate the function of the third domain of TFPI. By sequencing the gene, we will define the molecular basis of TFPI deficiency in patients who have been identified to contain 50% (or less) of normal plasma levels of TFPI. Both of them have had thrombotic episodes and are normal with respect to other known anticoagulant and fibrinolytic mechanisms. In other experiments, we will further examine whether or not the protease domain of factor IXa contains a part of the binding site for factor VIIIa. Likewise, whether or not the protease domain of factor Ixa also interact with factor VIIIa will be investigated. We will also test a hypothesis that the EGF-1 and/or EGF-2 domain of factor IX is needed for its activation by the tissue factor/factor VIIa complex. We anticipate that the information obtained from the proposed studies will significantly enhance our understanding about the molecular mechanisms involved in the assembly of intrinsic factor X-activating complex. In addition, the studies will define the molecular basis of TFPI deficiency in the patients investigated.